The present invention relates to electrophoretic separation of biomolecules, particularly to polyacrylamide medium to electrophoretically separate DNA sequencing extension products by capillary electrophoresis, and more particularly to high molecular weight polyacrylamides having a viscosity average molecular weight of around 675-725 kDa synthesized by conventional red-ox polymerization technique for electrophoretic separation of DNA or other biomolecules.
Capillary electrophoresis (CE) of DNA requires some type of non-charged polymer solution due to free-draining coil behavior of DNA. Addition of polymer to the separation medium changes, the frictional drag of DNA thereby making mobility of DNA a function of its size.
Polyacrylamide (PAAm) is one of the popular polymer in preparing separation medium for DNA sequencing by CE. The objective was to achieve high-resolution and long read-lengths in short run-times while keeping the solution viscosity to moderate levels. However, so far the objective is met with partial degree of success only. Furthermore, routine synthesis and uniform dilution of PAAm to a given final concentration is problematic and takes a long time. Grossman, J. Chromatograph A, 1994, 663, 219-227, reported the use of low molecular weight PAAm solution, but the PAAm needed to be isolated from the polymerization mixture before it was reconstituted to the final concentration.
Low concentrations of high molecular weight PAAm were used by Carrilho, E. et al., Anal. Chem., 1996, 68,3305-3313, and reported a read-length of xcx9c1000 bases at a resolution value of 0.1. However, the PAAm stock solution takes 2-3 days for uniform dilution to a final concentration, which therefore is inconvenient and time-consuming. Goetzinger, W., et al., Electrophoresis, 1998, 19, 242-248, reported the advantages of high molecular weight PAAm made by well known emulsion polymerization technique to make uniform dilution of PAAm more convenient. Further optimization of the separation media to increase the performance was attempted by Salas-Solano, O. et al., Anal. Chem., 1998, 70,4003,3996-4003, using a mixture of two different average molecular weights of PAAm and reported a read-length of xcx9c1000 bases (resolution value not reported). In both latter cases, PAAm needed to be separated and purified before reconstituting to a final concentration which makes the process less than ideal for production environment.
However, consistency in synthesizing high molecular weight PAAm is still a challenge. This problem compromises high-resolution and long read-lengths. To circumvent this problem, Solano et al referenced above, used PAAm synthesized from well known emulsion polymerization methods. However, this method is cumbersome and requires the separation and purification of PAAm from the polymerization mixture before it can be reconstituted. Furthermore, these prior methods also needed two different average molecular weight PAAm(s) to achieve the desired high-resolution.
The present invention provides a solution to the above referenced problems, and comprises a simple and consistent formulation or method of making very high molecular weight PAAm. Separation medium formulated from the polymer of this invention has yielded better resolution than the commercially supplied medium, such as that commercialized by Amersham Pharmacia (Molecular Dynamics, Sunnyvale, Calif.) under identical separation conditions. Here, we report a simple formulation of PAAm separation medium, based on a single average molecular weight PAAm synthesized from conventional red-ox polymerization method. The PAAm stock solution was made to uniform dilution in xcx9c24 hours to the final concentration. Additionally, this formulation has better performance than the commercially supplied PAAm medium from Amersham Pharmacia making it convenient and potentially cost-effective in a production sequencing environment. For example, the resolution achieved with the formulation of the present invention to separate DNA under identical conditions is much superior (725 bases vs. 625 bases) and faster (60 min. vs. 75 min.) to the commercially supplied product from Amersham Pharmacia. Using the present invention, high molecular weight PAAm having a viscosity average molecular weight (Mv) of 674 kDa were synthesized by conventional red-ox polymerization technique.
It is an object of the present invention to provide polyacrylamide medium for electrophoretic separation of biomolecules.
A further object of the invention is to enable synthesizing high molecular weight polyacrylamide for capillary electrophoresis.
A further object of the invention is to provide a simple and consistent method of making very high molecular weight polyacrylamide.
Another object of the invention is to provide high molecular weight polyacrylamides having a viscosity average molecular weight of around 675-725 kDa which are synthesized by conventional red-ox polymerization techniques.
Another object of the invention is to provide a high molecular weight polyacrylamide that provides a resolution for separate DNA that is superior (725 bases) and faster (60 min.) to the commercially available polyacrylamides.
Another object of the invention is to provide a method for producing high molecular weight polyacrylamides which is straight-forward, simple, and does not require cumbersome processing such as emulsion polymerization.
Another object of the invention is to provide a method for producing a polyacrylamide medium which does not require separation of the polyacrylamide from the reaction mixture prior to reconstituting the polymer to a final concentration.
Another object of the invention is to provide a polyacrylamide formulation which is prepared from a single average molecular weight polyacrylamide as opposed to the mixture of two different average molecular weight polyacrylamides required to achieve high resolution by prior known approaches.
Other objects and advantages of the present invention will become apparent from the following description. The present invention involves a high molecular weight polyacrylamide medium for the electrophoretic separation of biomolecules, such as DNA. In accordance with the present invention, high molecular weight polyacrylamides (PAAM) having a viscosity average molecular weight (Mv) of around 675-725 kDa were synthesized by a conventional red-ox polymerization technique. This is achieved by optimizing the concentrations of acrylamide monomer, ammonium persulfate (APS), and tetramethylithylenediamine (TEMED) and polymerizing in aqueous medium in presence of urea at sub-ambient temperatures after efficiently removing oxygen from the reaction mixture by bubbling ultra-pure argon. The resultant polymer solution is further diluted to a suitable final concentration with an electrophoretic buffer, such as TRIS-TAPS-EDTA (TTE). The methods employed here to synthesize PAAm is straight-forward, simple and does not require cumbersome methods such as emulsion polymerization in order to achieve very high molecular weights. The method here also does not require separation of PAAm from the reaction mixture prior to reconstituting the polymer to a final concentration.
Furthermore, the formulation is prepared from a single average mol. wt. PAAm as opposed to the mixture of two different average mol. wt. PAAm required to achieve high resolution, as reported in the above-referenced article by Solano et al. Thus, the present invention provides consistency in synthesizing high molecular weight PAAm, thereby providing a solution to the above-discussed problems of the prior known synthesizing approaches.